Analysis of the Role of Aminoacyl tRNA Synthetase Genes in Global Protein Synthesis and mRNA Specific Regulation of Translation in Cancer Cells

Analysis of the Role of Aminoacyl tRNA Synthetase Genes in Global Protein Synthesis and mRNA Specific Regulation of Translation in Cancer Cells Elyse Nguyen, Depts. of Biology and Chemistry, Dipak Poria, & Esta Sterneck, with Dr. Sarah Williams, Dept. of Forensic Science Coordinated control of transcription and translation of gene expression impels cellular fate decision under different microenvironmental stresses. Cancer cells often usurp these regulatory machineries to adapt under microenvironmental stress or under therapeutic intervention. The transcription factor CEBPδ is induced by various stressors and mediates cellular adaptation and survival. RNA-seq analysis of a CEBPD-silenced human melanoma cell line, MB-435s, showed decreased expression of 12 aminoacyl-tRNA synthetase (aaRS) genes. Our group recently found that deletion of CEBPD by CRISPR/Cas9 (CEBPD-KO) compromised aminoacyl tRNA synthetase (aaRS) expression and global protein synthesis. However, despite this decrease in global protein production, the synthesis of certain proteins, such as ATF4, which promotes survival and/or death under stress conditions, is increased. Aminoacyl tRNA synthetases are essential enzymes in the process of protein synthesis which catalyze the addition of appropriate amino acid to its corresponding tRNA, and therefore act as a rate limiting step in cellular protein synthesis. In the current project, we sought to investigate the effect of silencing of specific aaRS genes, glutamyl-prolyl-tRNA synthetase (EPRS) and valyl-tRNA synthetase (VARS) on global protein translation and ATF4 expression. To address this question, we silenced the EPRS and VARS gene expression using two independent short-hairpin-RNA (shRNA) targeting two different regions of EPRS and VARS mRNAs in MB-435s cells. Silencing of EPRS gene showed compensatory upregulation of VARS and vice versa. Interestingly, our preliminary data suggested an upregulation of global protein synthesis after EPRS and VARS silencing in MB435s cells measured by puromycin pulse labelling. Ongoing experiments to validate the preliminary data and ATF4 expression will be discussed.https://scholarscompass.vcu.edu/uresposters/1326/thumbnail.jp

Detection of pediatric upper extremity motor activity and deficits with accelerometry

Importance: Affordable, quantitative methods to screen children for developmental delays are needed. Motor milestones can be an indicator of developmental delay and may be used to track developmental progress. Accelerometry offers a way to gather real-world information about pediatric motor behavior. Objective: To develop a referent cohort of pediatric accelerometry from bilateral upper extremities (UEs) and determine whether movement can accurately distinguish those with and without motor deficits. Design, Setting, and Participants: Children aged 0 to 17 years participated in a prospective cohort from December 8, 2014, to December 29, 2017. Children were recruited from Ranken Jordan Pediatric Bridge Hospital, Maryland Heights, Missouri, and Washington University School of Medicine in St Louis, St Louis, Missouri. Typically developing children were included as a referent cohort if they had no history of motor or neurological deficit; consecutive sampling and matching ensured equal representation of sex and age. Children with diagnosed asymmetric motor deficits were included in the motor impaired cohort. Exposures: Bilateral UE motor activity was measured using wrist-worn accelerometers for a total of 100 hours in 25-hour increments. Main Outcomes and Measures: To characterize bilateral UE motor activity in a referent cohort for the purpose of detecting irregularities in the future, total activity and the use ratio between UEs were used to describe typically developing children. Asymmetric impairment was classified using the mono-arm use index (MAUI) and bilateral-arm use index (BAUI) to quantify the acceleration of unilateral movements. Results: A total of 216 children enrolled, and 185 children were included in analysis. Of these, 156 were typically developing, with mean (SD) age 9.1 (5.1) years and 81 boys (52.0%). There were 29 children in the motor impaired cohort, with mean (SD) age 7.4 (4.4) years and 16 boys (55.2%). The combined MAUI and BAUI (mean [SD], 0.86 [0.005] and use ratio (mean [SD], 0.90 [0.008]) had similar F1 values. The area under the curve was also similar between the combined MAUI and BAUI (mean [SD], 0.98 [0.004]) and the use ratio (mean [SD], 0.98 [0.004]). Conclusions and Relevance: Bilateral UE movement as measured with accelerometry may provide a meaningful metric of real-world motor behavior across childhood. Screening in early childhood remains a challenge; MAUI may provide an effective method for clinicians to measure and visualize real-world motor behavior in children at risk for asymmetrical deficits

The antisense oligonucleotide nusinersen for treatment of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a rare, autosomal recessive neuromuscular degenerative disease characterized by loss of spinal cord motor neurons leading to progressive muscle wasting. The most common pathology results from a homozygous disruption in the survival motor neuron 1 (SMN1) gene on chromosome 5q13 via deletion, conversion, or mutation. SMN2 is a near duplicate of SMN1 that can produce full-length SMN mRNA transcripts, but its overall production capability of these mRNA transcripts is lower than that seen in SMN1. This leads to lower levels of functional SMN protein within motor neurons. The FDA approved nusinersen in December 2016 to treat SMA associated with SMN1 gene mutation. It is administered directly to the central nervous system by intrathecal injection. An antisense oligonucleotide (ASO) drug, nusinersen, provides an upcoming and promising treatment option for SMA and represents a novel pharmacological approach with a mechanism of action relevant for other neurodegenerative disorders. Nusinersen begins with four initial loading doses that are followed by three maintenance doses per year. Three major studies (CHERISH, ENDEAR, and NURTURE) have shown to improve motor function in early and late-onset individuals and reduce the chances of ventilator requirements in pre-symptomatic infants. Studies investigating the timing of drug delivery in mouse models of SMA report the best outcomes when drugs are delivered early before any significant motor function is lost. Nusinersen is a novel therapeutic approach with consistent results in all three studies and is proof of the novel concept for treating SMA and other neurodegenerative disorders in the future

Enhancer viruses and a transgenic platform for combinatorial cell subclass-specific labeling

The rapid pace of cell type identification by new single-cell analysis methods has not been met with efficient experimental access to the newly discovered types. To enable flexible and efficient access to specific neural populations in the mouse cortex, we collected chromatin accessibility data from individual cells and clustered the single-cell data to identify enhancers specific for cell classes and subclasses. When cloned into adeno-associated viruses (AAVs) and delivered to the brain by retro-orbital injections, these enhancers drive transgene expression in specific cell subclasses in the cortex. We characterize several enhancer viruses in detail to show that they result in labeling of different projection neuron subclasses in mouse cortex, and that one of them can be used to label the homologous projection neuron subclass in human cortical slices. To enable the combinatorial labeling of more than one cell type by enhancer viruses, we developed a three-color Cre-, Flp- and Nigri- recombinase dependent reporter mouse line, Ai213. The delivery of three enhancer viruses driving these recombinases via a single retroorbital injection into a single Ai213 transgenic mouse results in labeling of three different neuronal classes/subclasses in the same brain tissue. This approach combines unprecedented flexibility with specificity for investigation of cell types in the mouse brain and beyond

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome

Simulated quantum sampling of sloan optotypes

PURPOSE The purpose of this experiment was to simulate, under photopic conditions, quantum spatial sampling of Sloan optotypes, to determine the minimum number of randomly positioned spatial samples required by observers to identify such letters. METHODS Subjects were the study's authors. We stimulated quantum sampling of Sloan optotypes by presenting them on a computer monitor, with varied probabilities of individual pixels being switched on. The threshold pixel probability was measured using a descending staircase, with optotypes being presented in blocks of 5 at a given pixel probability, with pixel probability being decreased by 0.2 log units on successive blocks, until more than 3 mistakes were made on a given block. Bailey's letter counting procedure, and also Probit analysis, was used to determine threshold pixel probability. Threshold pixel probabilities were measured for letter sizes ranging from letter sizes ranging from 20/40 to 20/2560, for negative contrast (traditional dark letters on a bright background), and for positive contrast (bright letters on a dark background) and for different simulated Weber contrasts (by adjusting the probability of pixels being switched on in dark areas). RESULTS For maximum contrast letters, threshold pixel probability varied widely with letter size, in an inverse square relationship. However the threshold number of pixels required for letter identification was constant for a wide variety of letter sizes. For positive contrast stimuli, letters were identifiable when a threshold average of 14 to 16 pixels were presented at random positions on the letter surface. For negative contrast stimuli, 25 to 30 randomly distributed pixels had to be blocked by a letter for threshold identification. Simulating decreased contrast, by even small amounts, significantly elevated identification thresholds. For 50 % simulated Weber contrast, 500 randomly distributed pixels were required to identify positive or negative contrast stimuli. CONCLUSIONS Our results, may assist in understanding the quantum and background-noise limitations on vision at very low light levels

The antisense oligonucleotide nusinersen for treatment of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a rare, autosomal recessive neuromuscular degenerative disease characterized by loss of spinal cord motor neurons leading to progressive muscle wasting. The most common pathology results from a homozygous disruption in the survival motor neuron 1 (SMN1) gene on chromosome 5q13 via deletion, conversion, or mutation. SMN2 is a near duplicate of SMN1 that can produce full-length SMN mRNA transcripts, but its overall production capability of these mRNA transcripts is lower than that seen in SMN1. This leads to lower levels of functional SMN protein within motor neurons. The FDA approved nusinersen in December 2016 to treat SMA associated with SMN1 gene mutation. It is administered directly to the central nervous system by intrathecal injection. An antisense oligonucleotide (ASO) drug, nusinersen, provides an upcoming and promising treatment option for SMA and represents a novel pharmacological approach with a mechanism of action relevant for other neurodegenerative disorders. Nusinersen begins with four initial loading doses that are followed by three maintenance doses per year. Three major studies (CHERISH, ENDEAR, and NURTURE) have shown to improve motor function in early and late-onset individuals and reduce the chances of ventilator requirements in pre-symptomatic infants. Studies investigating the timing of drug delivery in mouse models of SMA report the best outcomes when drugs are delivered early before any significant motor function is lost. Nusinersen is a novel therapeutic approach with consistent results in all three studies and is proof of the novel concept for treating SMA and other neurodegenerative disorders in the future

OVOL1 Regulates Psoriasis-Like Skin Inflammation and Epidermal Hyperplasia.

Psoriasis is a common inflammatory skin disease characterized by aberrant inflammation and epidermal hyperplasia. Molecular mechanisms that regulate psoriasis-like skin inflammation remain to be fully understood. Here, we show that the expression of Ovol1 (encoding ovo-like 1 transcription factor) is upregulated in psoriatic skin, and its deletion results in aggravated psoriasis-like skin symptoms following stimulation with imiquimod. Using bulk and single-cell RNA sequencing, we identify molecular changes in the epidermal, fibroblast, and immune cells of Ovol1-deficient skin that reflect an altered course of epidermal differentiation and enhanced inflammatory responses. Furthermore, we provide evidence for excessive full-length IL-1α signaling in the microenvironment of imiquimod-treated Ovol1-deficient skin that functionally contributes to immune cell infiltration and epidermal hyperplasia. Collectively, our study uncovers a protective role for OVOL1 in curtailing psoriasis-like inflammation and the associated skin pathology